Limb loss is a major health concern in the U.S. with nearly two million patients living with the consequences of a major limb amputation. This number is expected to rise with increases in key risk factors, and no biological therapeutics has been devised to address this problem. While humans have exceedingly limited regenerative abilities in limbs and other key structures, axolotl salamanders can regenerate entire limbs throughout their lives. Axolotl limbs are anatomically similar to human limbs, and they develop by similar mechanisms. Gaining a thorough understanding of the molecular mechanisms that enable axolotl limb regeneration stands to offer critical insights into future approaches that may be taken in regenerative medicine, which could in turn revolutionize the treatment options offered to patients facing amputation. This thorough mechanistic understanding has evaded researchers to date because of a paucity of tools available for experimentally manipulating gene expression in axolotls. However, within the last eight years, we-and others-have developed powerful molecular genetic tools that are operational in vivo in axolotls. We propose to leverage these developments to take a fresh look at the longstanding and important question of vertebrate limb regeneration. In axolotls, one of the earliest events post-amputation is the formation of a specialized wound epidermis across the stump. Beneath this wound epidermis, progenitor cell pool for internal tissues, the blastema, forms. Blastemas are critical for limb regeneration, but their creation and growth are poorly understood, and there is a strong possibility that both aspects are under the control of the wound epidermis. Precise roles for wound epidermis and its molecular factors have been elusive because of a lack of tools for studying these questions to date. Through a massive RNA-sequencing effort extended to single-cell level, we have identified candidate genes whose expression is highly enriched in the wound epidermis versus blastema cells and all other tissues sampled. Here we propose to leverage this data set as well as our recently-developed retrovirus system for infecting axolotls in vivo to answer specific questions about the role of wound epidermis and to examine five specific genes. In Specific Aim 1, we will determine if wound epidermis is required for activation of cartilage and muscle progenitors post-amputation using a retrovirus to mark activated cells. In Specific Aim 2, we will test the sufficiency of five wound-epidermis-enriched genes to cause dedifferentiation or stem cell activation in limbs without wound epidermis. These experiments will allow us to establish a system whereby we can study critical cellular events downstream of the wound epidermis, and it test the feasibility of using this approach to identify key molecular components of wound epidermis. Performing this research enable future experiments aimed at more intensive dissection of the molecular pathways that support the wound epidermis functions, it and will lay the groundwork for considering the role of these processes in the mammalian context.